Single-molecule imaging provides a method for the direct observation of polyomavirus infection that is not possible using conventional, ensemble, techniques. The infection mechanisms of the polyomaviruses, with the exception of SV40, are not well-understood. It has been suggested that the murine polyoma virus is internalized independently of clathrin or caveolae. We propose to use single-molecule imaging techniques to characterize the internalization and endocytic pathways of the murine polyoma virus and the human polyomavirus, JCV. Single-molecule imaging studies of MPV and JCV will characterize the broad range of endocytic pathways necessary for viral infection. Comparisons to SV40 and influenza will provide a broad understanding of viral infection that will be essential in the development of antiviral drugs.